Method for Expanding Monocytes

ABSTRACT

The invention relates to an ex vivo method for expanding monocytes, macrophages or dendritic cells, which method comprises inhibiting the expression or the activity of MafB and c-Maf in monocytes, macrophages or dendritic cells; and expanding the cells in the presence of at least one cytokine or an agonist of cytokine receptor signaling.

The invention relates to a method for generating, maintaining andexpanding monocytes, macrophages or dendritic cells in long termculture.

BACKGROUND OF THE INVENTION

Monocytes are generated in the bone marrow (BM) to be released in theblood stream and give rise to different types of tissue-macrophagesordendritic cells after leaving the circulation. Monocytes, theirprogeny and immediate precursors in the bone marrow have also been namedthe ‘mono-nuclear phagocyte system’ (MPS). They are derived fromgranulocyte/macrophage colony forming unit (CFU-GM) progenitors in thebone marrow that gives rise to monocytic and granulocytic cells. Thematuration process of the monocytic lineage in vivo passes from amonoblast stage, through the promonocyte stage to mature monocytes (GoudT J et al. 1975). IL-3, GM-CSF and macrophage colony stimulating factor(M-CSF) stimulate in vivo generation of monocytes (Metcalf D et al.1990). In vitro, hematopoietic progenitor cells cultured with GM-CSFinduce CFU-GM to differentiate towards granulocytes, while addition ofFL and SCF shifts differentiation from the granulocytic to the monocyticlineage (Gabbianelli M et al. 1995; Willems R et al. 2001).

Several recent studies indicate that although specific surface markerexpression may vary in detail between species, the generaldifferentiation pathways of monocytes and their progeny appear to belargely conserved between mice and humans (Gordon S et al. 2005). Withthe higher accessibility to experimentation a more detaileddifferentiation pathway could be worked out in the animal model. Mousemonocytes originate from hematopoietic stem cells (HSC) in the bonemarrow via successive commitment steps and several intermediateprognitor stages with increasingly restricted differentiation potential(Shizuru J A et al. 2005; Kondo M et al. 2000). This differentiationseries is believed to pass through the common myeloid progenitor (CMP),which can give rise to all myeloid cells, to the granulocyte-macrophageprogenitor (GMP), which gives rise to monocytic and granulocytic cellsand may be identical or very similar to CFU-GM progenitors. Yet a moreimmediate monocytic progenitor in the bone marrow appears to be themacrophage/dendritic cell progenitor (MDP) that can give rise tomacrophages and dendritic cells, likely via a monocyte intermediatestage (Fogg et al 2006).

Newly formed monocytes leave the BM within 24 hours and migrate to theperipheral blood. Circulating monocytes can adhere to endothelial cellsof the capillary vessels and are able to migrate into various tissues(van Furth R. et al. 1992), where they can differentiate intomacrophages or dendritic cells. These adherence and migration involvesurface proteins, lymphocyte-function associated antigen-1 (LFA-1), CD11and antigen-4 (VLA-4), belonging to the integrin superfamily of adhesionmolecules (Kishimoto T K et al. 1989). These integrins interact withselectins on endothelial cells. Monocyte derived macrophages can show ahigh degree of heterogeneity that reflects a morphological andfunctional specification adopted in the infiltrated tissue. According totheir anatomical localization they may also have distinct names (e.g.microglia in the central nervous system and Kupffer cells in the liver).

Although it remains controversial, whether some resident macrophagepopulations may be capable of proliferating in situ under certainconditions, the majority of macrophages appear to have no or a verylimited proliferation capacity (Gordon S, et al., 2005). The renewal oftissue macrophage populations therefore depends on the influx ofmonocytes and their local differentiation (Crofton R W et al. 1978;Blusse van Oud Alblas A et al; 1981). Although such tissue infiltratingmonocytes can have a very limited proliferation ability, monocytescirculating in the blood do not cycle and rapidly differentiate intomacrophages rather than expand, when stimulated with M-CSF ex vivo.

Monocyte recruitment to tissues differs under homeostatic andinflammatory conditions and appears to involve two distinct monocytepopulations that have been identified in humans and mammalian animalmodels (Gordon S, et al. 2005). During inflammation monocytopoiesisincreases (Shum D T et al. 1982; van Waarde D et al. 1977) resulting inelevated monocyte numbers. Furthermore, inflammatory mediators, IL-1,IL-4, IFN-γ and TNF-α upregulate expression of selectins on endothelialcells, promoting migration of monocytes into tissues. The same cytokinesmodulate expression of integrin adhesion molecules on monocytes (Pober JS et al. and 1990). At the site of inflammation monocytes are involvedin the phagocytosis of opsonized microorganisms or immune complexes viasurface γ receptors (CD64, CD32) and complement receptors (CD11b,CD11c). The microorganisms are synergistically killed by reactive oxygenand nitrogen metabolites and through several hydrolytic enzymes (acidphosphatase, esterase, lysozyme and galactosidase) (Kuijpers T. 1989;Hibbs J B et al. 1987). Importantly, monocyte derived macrophages anddendritic cells stimulate T cells by antigen presentation and thus, areinvolved in the recognition and activation phases of adaptive immuneresponses (Nathan C F. 1987). Monocytes also secrete a large number ofbioactive products which play an important role in inflammatory,proliferative and immune responses, including growth factors (GM-CSF,G-CSF, M-CSF, IL-1) and antiproliferating factors (IFNs, TNF).

Lipopolysaccharide (LPS) or endotoxin is a predominant integralstructural component of the outer membrane of Gram-negative bacteria andone of the most potent microbial initiators of inflammation. LPS bindsto the CD14 glycoprotein that is expressed on the surface of monocytesand stimulates the toll receptor pathway via activation of TLR4. OtherPAMPs (pathogen associated molecular patterns) can also initiateinflammatory responses via other TLR receptors. The binding of LPS orother PAMPS induces production of TNF-α, IL-1, -6, -8 and -10 (Wright SD. Et al; 1990; Dobrovolskaia M A et al. 2002; Foey A D. et al. 2000).

Other than LPS or other PAMPs, one of the most efficient stimuli forcytokine production in vitro is the direct cell-cell contact ofmonocytes with activated lymphocytes (Wey E. et al. 1992; Parry S L. Etal. 1997), via CD40 ligand (CD40L) (Wagner D H. Et al. 1997; Shu U. etal. 1995; Alderson M R. et al. 1993). This interaction may also beimportant in the immune surveillance of tumors. Thus the incubation ofmonocytes with CD40L-transfected cells results in tumoricidal activityagainst a human melanoma cell line. Furthermore functional interactionshave also been described between monocytes and NK cells, a cell typewith significant anti-tumor activity. Both direct cell-cell contact(Miller J S. Et al. 1992). and release of soluble factors such as IL-12,TNF-α, IL-15 or IL-1β by activated monocytes induce proliferation,production of IFN-γ (Carson W E et al. 1995; Tripp C S. et al. 1993) andthe cytotoxic potential of cocultured NK cells in a time dependentmanner (Chang Z L. et al. 1990; Bloom E T. et al. 1986).

Finally macrophages are also critically involved in woundhealing andtissue repair, where they assume trophic functions by removing debrisand orchestrating the recruitment and activity of other cell typesparticipating in tissue remodelling (Gordon S et al. 2003)

Dendritic cells (DCs) are components of the innate immune system. Theyare antigen presenting cells with the unique ability to induce a primaryimmune response (Banchereu et al. 2000). They can be derived fromcirculating monocytes or circulating DC progenitors in the blood andnon-lymphoid peripheral tissues, where they can become resident cells(Bancherreau J. et al. 1998, 2000) (Geissmann, 2007) (Wu and Liu, 2007).Immature DCs (iDCs) recognize pathogens through cell surface receptors,including Toll-like receptors (Reis e Sousa C. 2001). After uptake ofantigen DCs mature and migrate to lymph nodes. Mature DCs (mDCs) areefficient antigen presenting cells (APCs) which mediate T cell priming(Banchereau J. et al. 1998, 2000). Furthermore a predominant role of DCshas been described in NK cell activation in mice and humans. Bothimmature and bacterially activated human monocyte-derived DCs have beenshown to induce cytokine secretion and cytotoxicity by NK cells(Ferlazzo G. et al. 2002; Fernandez N C. et al. 1999).

The in vitro differentiation of murine macrophages from bone marrow inthe presence of M-CSF was described by Stanley et al. (1978, 1986).Whereas progenitor cells will initially proliferate in response toM-CSF, they eventually differentiate to mature macrophages andterminally withdraw from the cell cycle (Pixley and Stanley, 2004). Thuseven though the macrophages generated this way will survive for alimited time, they are not homogeneous and cannot be further expanded inculture. Similarly human monocytes do not proliferate in response toM-CSF but initiate morphological changes indicative of macrophagedifferentiation (Becker et al., 1987). Although a significant number ofmonocytes can be obtained from a patient by leukapheresis andelutriation (Stevenson et al., 1983), these cells will furtherdifferentiate to macrophages in a few days without proliferating andcannot be maintained in culture.

Now, the present invention provides a new in vitro method forgenerating, maintaining and expanding monocytes and macrophages in longterm culture.

The inventors have indeed demonstrated that it is possible to expand andmaintain monocytes and macrophages in culture for weeks or months, byinactivating the expression of MafB and c-Maf in said cells. Not only invitro generated macrophages but also mature bone marrow MafB and c-Mafdeficient macrophages and blood monocytes continue to proliferate inculture.

Methods of the invention may thus be useful for therapeutic approachesrequiring amplification of monocytes and monocyte derived cells, as wellas for screening for drugs targeting monocyte, monocyte derivedmacrophages (including osteoclasts) and dendritic cells or for testingthe response to specific drugs in a patient specific way, or forstudying the molecular basis of monocyte or monocyte derived celldependent diseases by culturing and expanding monocyte or monocytederived cells of afflicted patients.

SUMMARY OF THE INVENTION

The present invention provides an ex vivo method for expandingmonocytes, macrophages or dendritic cells, which method comprisesinhibiting the expression or the activity of MafB and c-Maf inmonocytes, macrophages or dendritic cells; and expanding the cells inthe presence of at least one cytokine, e.g. M-CSF.

An object the invention is also a monocyte, macrophage or dendritic cellobtainable by the above method.

Another object of the invention is a monocyte, macrophage or dendriticcell, which does not express MafB and c-Maf or in which the expressionor activity of MafB and c-Maf is abolished or inhibited.

Such monocyte, macrophages or dendritic cells are useful in apharmaceutical composition, where they are in combination with apharmaceutically acceptable carrier.

A particular object of the invention is a pharmaceutical compositionwhich comprises such dendritic cell, loaded with an antigenic molecule,for use as a vaccine.

The invention further provides the use of a monocyte, macrophage ordendritic cell as defined above, for the screening of drugs.

The invention further provides a method for generating and expandingmurine monocytes, which method comprises the steps consisting of:

i) isolating monocytes derived from a mouse which does not express MafBand c-Maf and

ii) culturing said monocytes in the presence of M-CSF.

DETAILED DESCRIPTION Definitions

A “coding sequence” or a sequence “encoding” an expression product, suchas a RNA, polypeptide, protein, or enzyme, is a nucleotide sequencethat, when expressed, results in the production of that RNA,polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodesan amino acid sequence for that polypeptide, protein or enzyme. A codingsequence for a protein may include a start codon (usually ATG) and astop codon.

The term “gene” means a DNA sequence that codes for or corresponds to aparticular sequence of amino acids which comprise all or part of one ormore proteins or enzymes, and may or may not include regulatory DNAsequences, such as promoter sequences, which determine for example theconditions under which the gene is expressed. A “promoter” or “promotersequence” is a DNA regulatory region capable of binding RNA polymerasein a cell and initiating transcription of a downstream (3′ direction)coding sequence. Some genes, which are not structural genes, may betranscribed from DNA to RNA, but are not translated into an amino acidsequence. Other genes may function as regulators of structural genes oras regulators of DNA transcription. In particular, the term gene may beintended for the genomic sequence encoding a protein, i.e. a sequencecomprising regulator, promoter, intron and exon sequences.

As used herein, references to specific proteins (e.g., MafB or c-Maf)can include a polypeptide having a native amino acid sequence, as wellas variants and modified forms regardless of their origin or mode ofpreparation. A protein that has a native amino acid sequence is aprotein having the same amino acid sequence as obtained from nature(e.g., a naturally occurring MafB or c-Maf). Such native sequenceproteins can be isolated from nature or can be prepared using standardrecombinant and/or synthetic methods. Native sequence proteinsspecifically encompass naturally occurring truncated or soluble forms,naturally occurring variant forms (e.g., alternatively spliced forms),naturally occurring allelic variants and forms includingpostranslational modifications. A native sequence protein includesproteins following post-translational modifications such asglycosylation, or phosphorylation, ubiquitination, sumoylation or othermodifications of some amino acid residues.

The terms “mutant” and “mutation” mean any detectable change in geneticmaterial, e.g. DNA, RNA, cDNA, or any process, mechanism, or result ofsuch a change. This includes gene mutations, in which the structure(e.g. DNA sequence) of a gene is altered, any gene or DNA arising fromany mutation process, and any expression product (e.g. protein orenzyme) expressed by a modified gene or DNA sequence. Mutations includedeletion, insertion or substitution of one or more nucleotides. Themutation may occur in the coding region of a gene (i.e. in exons), inintrons, or in the regulatory regions (e.g. enhancers, responseelements, suppressors, signal sequences, polyadenylation sequences,promoters) of the gene. Generally a mutation is identified in a subjectby comparing the sequence of a nucleic acid or polypeptide expressed bysaid subject with the corresponding nucleic acid or polypeptideexpressed in a control population. Where the mutation is within the genecoding sequence, the mutation may be a “missense” mutation, where itreplaces one amino acid with another in the gene product, or a “nonsense” mutation, where it replaces an amino acid codon with a stopcodon. A mutation may also occur in a splicing site where it creates ordestroys signals for exon-intron splicing and thereby lead to a geneproduct of altered structure. A mutation in the genetic material mayalso be “silent”, i.e. the mutation does not result in an alteration ofthe amino acid sequence of the expression product.

Variants refer to proteins that are functional equivalents to a nativesequence protein that have similar amino acid sequences and retain, tosome extent, one or more activities of the native protein. Variants alsoinclude fragments that retain activity. Variants also include proteinsthat are substantially identical (e.g., that have 80, 85, 90, 95, 97,98, 99%, sequence identity) to a native sequence. Such variants includeproteins having amino acid alterations such as deletions, insertionsand/or substitutions. A “deletion” refers to the absence of one or moreamino acid residues in the related protein. The term “insertion” refersto the addition of one or more amino acids in the related protein. A“substitution” refers to the replacement of one or more amino acidresidues by another amino acid residue in the polypeptide. Typically,such alterations are conservative in nature such that the activity ofthe variant protein is substantially similar to a native sequenceprotein (see, e.g., Creighton (1984) Proteins, W.H. Freeman andCompany). In the case of substitutions, the amino acid replacing anotheramino acid usually has similar structural and/or chemical properties.Insertions and deletions are typically in the range of 1 to 5 aminoacids, although depending upon the location of the insertion, more aminoacids can be inserted or removed. The variations can be made usingmethods known in the art such as site-directed mutagenesis (Carter, etal. (1985); Zoller et al. (1982) Nucl. Acids Res. 10:6487), cassettemutagenesis (Wells et al. (1985) Gene 34:315), restriction selectionmutagenesis (Wells, et al. (1986) Philos. Trans. R. Soc. London SerA317:415), and PCR mutagenesis (Sambrook et al., 2001).

Two amino acid sequences are “substantially homologous” or“substantially similar” when greater than 80%, preferably greater than85%, preferably greater than 90% of the amino acids are identical, orgreater than about 90%, preferably grater than 95%, are similar(functionally identical). Preferably, the similar or homologoussequences are identified by alignment using, for example, the GCG(Genetics Computer Group, Program Manual for the GCG Package, Version 7,Madison, Wis.) pileup program, or any of sequence comparison algorithmssuch as BLAST, FASTA, etc.

The term “expression” when used in the context of expression of a geneor nucleic acid refers to the conversion of the information, containedin a gene, into a gene product. A gene product can be the directtranscriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisenseRNA, ribozyme, structural RNA or any other type of RNA) or a proteinproduced by translation of a mRNA. Gene products also include messengerRNAs which are modified, by processes such as capping, polyadenylation,methylation, and editing, and proteins (e.g., MafB or c-Maf) modifiedby, for example, methylation, acetylation, phosphorylation,ubiquitination, SUMOylation, ADP-ribosylation, myristilation, andglycosylation.

An “inhibitor of expression” refers to a natural or synthetic compoundthat reduces or suppresses the expression of a gene.

An “inhibitor of activity” has its general meaning in the art, andrefers to a compound (natural or not) which has the capability ofreducing or suppressing the activity of a protein.

The term “c-Maf” denotes the c-Maf proto-onocogene, which is identicalin sequence to the v-Maf oncogene of AS42 virus and can transformchicken embryo fibroblasts (Nishizawa et al. PNAS 1989). C-Maf and otherMaf family members form homodimers and heterodimers with each other andwith Fos and Jun, consistent with the known ability of the AP-1 proteinsto pair with each other (Kerppola, T. K. and Curran, T. (1994) Oncogene9:675-684; Kataoka, K. et al. (1994) Mol. Cell. Biol. 14:700-712). TheDNA target sequence to which c-Maf homodimers bind, termed the c-Mafresponse element (MARE), is a 13 or 14 bp element which contains a coreTRE (T-MARE) or CRE (C-MARE) palindrome respectively, but c-Maf may alsobind to DNA sequences diverging from these consensus sites includingcomposite AP-1/MARE sites and MARE half sites with 5′ AT rich extensions(Yoshida et al., NAR 2005). c-Maf has been shown to stimulatetranscription from several promoters, including the Purkinjeneuron-specific promoter L7 (Kurscher, C. and Morgan, J. I. (1994) Mol.Cell. Biol. 15:246-254), α,β γ-Crystallin (Ring et al. Development,2000, Kim et al. PNAS 1999; Kawauchi et al. JBC 1999, Yang et al., JMB2005), insulin (Matsuoka, et al. MCB, 2003) and p53 (Hale et al., JBC2000) promoters as well as to repress the transcription of otherpromoters such as the early myeloid promoter AND/CD13 (Hedge et al.,1998). c-Maf has also been shown to induce the differentiation of Thelper 2 (Th2) cells (Ho et al., 1996) due to its ability to activatethe tissue specific transcription of the interleukin-4 (IL-4) (Kim etal. 1999). Furthermore the over-expression of c-Maf in myeloid celllines induces macrophage differentiation (Hegde et al., 1999). Thenucleotide sequence of the mouse c-maf proto-oncogene, and predictedamino acid sequence for the mouse c-Maf protein, have been described(Kurscher, C. and Morgan, J. I. (1995) Mol. Cell. Biol. 15:246-254; andGenbank Accession number S74567). The nucleotide sequence of the chickenc-maf proto-oncogene, and predicted amino acid sequence for the chickenc-Maf protein, also have been described (Kataoka et al. 1994, GenbankAccession number D28596). The nucleotide sequence of the human c-mafproto-oncogene, and predicted amino acid sequence for the human c-Mafprotein, also have been described (U.S. Pat. No. 6,274,338 and GenbankAccession number BD106780)

The term “MafB” denotes the MafB transcription factor. This gene isexpressed in a variety of cell types (including lens epithelial,pancreas endocrine, chondrocyte, neuronal and hematopoietic cells) andencodes a protein of 311 amino acids containing a typical bZip motif inits carboxy-terminal region. In the bZip domain, MafB shares extensivehomology not only with v/c-Maf but also with other Maf-related proteins.MafB can form a homodimer through its leucine repeat structure andspecifically binds Maf-recognition elements (MAREs) palindromes,composite AP-1/MARE sites or MARE halfsites with AT rich 5′ extensions(Yoshida, et al. 2005). In addition, MafB can form heterodimers withc-/v-Maf or Fos through its zipper structure but not with Jun or otherMaf family members (Kataoka et al., 1994). MafB is also known under thename kreisler, kr or Krml1 (for ‘Kreisler Maf leucine Zipper 1’),because an x-ray induced chromosomic micro-inversion in kreisler mutantmice causes the tissue specific loss of MafB expression in thedeveloping hindbrain that is responsible for the kreisler phenotype(Cordes et al., 1994) (Eichmann et al., 1997). In the hematopoieticsystem MafB is expressed selectively in the myeloid lineage and isup-regulated successively during myeloid differentiation frommultipotent progenitors to macrophages. Indeed, this induction reflectsan important role of MafB in monocytic differentiation. Thus theoverexpression of MafB in transformed chicken myeloblasts (Kelly et al.,2000, Bakri et al. 2005) and in human hematopoetic progenitors (Gemelliet al., 2006) inhibits progenitor proliferation (Tillmanns et al., 2007)and stimulates the rapid formation of macrophages (Kelly et al., 2000,Bakri et al. 2005, Gemelli et al., 2006), whereas a dominant negativeversion of MafB inhibits this process (Kelly et al., 2000), indicatingthat MafB induction is a specific and important determinant of themonocytic program in hematopoietic cells. The nucleotide sequence of thechicken (Kataoka, K. et al. 1994), mouse (Cordes et al. 1994) and human(Wang et al. 1999) MafB gene, and predicted amino acid sequences for theMafB proteins, also have been described (GenBank accession numbersNM_(—)001030852 (gallus gallus), NM_(—)010658 (mus musculus),NM_(—)005461 (homo sapiens) D28600).

A “monocyte cell” is a large mononuclear phagocyte of the peripheralblood. Monocytes vary considerably, ranging in size from 10 to 30 μm indiameter. The nucleus to cytoplasm ratio ranges from 2:1 to 1:1. Thenucleus is often band shaped (horseshoe), or reniform (kindey-shaped).It may fold over on top of itself, thus showing brainlike convolutions.No nucleoli are visible. The chromatin pattern is fine, and arranged inskein-like strands. The cytoplasm is abundant and appears blue gray withmany fine azurophilic granules, giving a ground glass appearance inGiemsa staining. Vacuoles may be present. More preferably, theexpression of specific surface antigens is used to determine whether acell is a monocyte cell. The main phenotypic markers of human monocytecells include CD11b, CD11c, CD33 and CD115. Generally, human monocytecells express CD9, CD11b, CD11c, CDw12, CD13, CD15, CDw17, CD31, CD32,CD33, CD35, CD36, CD38, CD43, CD49b, CD49e, CD49f, CD63, CD64, CD65s,CD68, CD84, CD85, CD86, CD87, CD89, CD91, CDw92, CD93, CD98, CD101,CD102, CD111, CD112, CD115, CD116, CD119, CDw121b, CDw123, CD127,CDw128, CDw131, CD147, CD155, CD156a, CD157, CD162, CD163, CD164, CD168,CD171, CD172a, CD180, CD206, CD131a1, CD213a2, CDw210, CD226, CD281,CD282, CD284, CD286 and optionally CD4, CD14, CD16, CD40, CD45RO,CD45RA, CD45RB, CD62L, CD74, CD142 and CD170, CD181, CD182, CD184,CD191, CD192, CD194, CD195, CD197, CX3CR1. The main phenotypic markersof mouse monocyte cells include CD11b+, CD115, F4/80+. Generally mousemonocytes express CD11a, CD11b, CD16, CD18, CD29, CD31, CD32, CD44,CD45, CD49d, CD115, CD116, Cdw131, CD281, CD282, CD284, CD286, F4/80,and optionally CD49b, CD62L, CCR2, CX3CR1, and Ly6C. Upon contact withsensitive target cells, monocyte cells also produce a number ofcytokines, including IFNs, TNFs, GM-CSF, G-CSF, M-CSF, and IL-1.

A “macrophage cell” is a cell exhibiting properties of phagocytosis. Themorphology of macrophages varies among different tissues and betweennormal and pathologic states, and not all macrophages can be identifiedby morphology alone. However, most macrophages are large cells with around or indented nucleus, a well-developed Golgi apparatus, abundantendocytotic vacuoles, lysosomes, and phagolysosomes, and a plasmamembrane covered with ruffles or microvilli. The key functions ofmacrophages in innate and adaptive immunity are the phagocytosis andsubsequent degradation of senescent or apoptotic cells, microbes andneoplastic cells, the secretion of cytokines, chemokines and othersoluble mediators, and the presentation of foreign antigens (peptides)on their surface to T lymphocytes. Macrophages are derived from commonmyeloid progenitor cells and granulocyte-monocyte progenitor cells inthe bone marrow of mammalian organisms, which ultimately develop throughfurther progenitor stages into monocytes that then enter the peripheralbloodstream. Unlike neutrophils, with their multilobed nuclei, monocyteshave kidney-shaped nuclei and assume a large cell body during furtherdifferentiation and activation. Throughout life, some monocytes adhereto and migrate through the endothelium of the capillaries into allorgans, where they differentiate into resident tissue macrophages ordendritic cells (see below). Besides a monocyte origin a limited selfrenewal capacity has also been reported for some subpopulations oftissue macrophages. Lymphatic tissues, such as the lymph nodes and thespleen, are particularly rich in macrophages. In some organs themacrophages carry special names, as summarized in Table 1.

TABLE 1 Examples of tissue macrophages Organ Macrophage population BoneOsteoclasts Central nervous system Microglia Connective tissueHistiocytes Chorion villi of the placenta Hofbauer cells KidneyMesangial cells Liver Kupffer cells Peritoneal cavity Peritonealmacrophages Pulmonary airways Alveolar macrophages Skin Epidermal anddermal macrophages Spleen Marginal zone macrophages, Metallophilicmacrophages, Red pulp macrophages, White pulp macrophages

In the context of the invention, the macrophage is selected from thegroup consisting of microglia, histiocytes, Hofbauer cells, mesangialcells, Kupffer cells, peritoneal macrophages, alveolar macrophage,epidermal or dermal macrophages, marginal zone macrophages,metallophilic macrophages, Red pulp macrophages, white pulp macrophagesand osteoclasts. Bone marrow or fetal liver derived macrophages areparticularly useful.

Osteoclasts are a specialized cell type of the mononuclear phagocytesystem that is specific to bone and serves an important homeostatic andremodelling function in this tissue by degrading its mineralizedcomponents. In culture, osteoclasts can be derived from CFU-GMprogenitors of the bone marrow and from blood monocytes by culture inM-CSF and RANKL. The importance of these cytokines for osteoclastdevelopment is underscored by osteoclast deficiency and development ofosteopetrosis in mice with deletions of either of these two factors.Although it has not been shown formally in vivo, it is widely assumedthat circulating blood monocytes serve as osteoclast precursors.Aberrant osteoclast development and/or activity play a prominent role indebilitating human pathologies of high prevalence and with limitedtreatment options, such as osteoporosis, osteopetrosis andosteoarthritis (Boyle W J et al. 2003; Teitelbaum S L et al. 2003).

Macrophages are an important source of cytokines. Functionally, thenumerous products can be placed into five major groups: (1) cytokinesthat mediate a proinflammatory response, i.e. help to recruit furtherinflammatory cells (e.g. IL-1, Il-6, TNFs, CC and CXC chemokines, suchas IL-8 and monocyte-chemotactic protein 1); (2) cytokines that mediateT cell and natural killer (NK) cell activation (e.g. IL-1, IL-12,IL-18); (3) cytokines that exert a feedback effect on the macrophageitself (e.g. IL-1, TNFs, IL-12, IL-18, M-CSF, IFNα/β, IFNγ); (4)cytokines that downregulate the macrophage and/or help to terminate theinflammation (e.g. IL-10, TGFβs), (5) cytokines important for woundhealing (e.g. EGF, PDGF, bFGF, TGFβ). The production of cytokines bymacrophages can be triggered by microbial products such as LPS, byinteraction with type 1 T-helper cells, or by soluble factors includingprostaglandins, leukotrienes and, most importantly, other cytokines(e.g. IFNγ). Generally, human macrophages express CD11c, CD11b, CD18,CD26, CD31, CD32, CD36, CD45R0, CD45RB, CD63, CD68, CD71, CD74, CD87,CD88, CD101, CD119, CD121b, CD155, CD156a, CD204, CD206 CDw210, CD281,CD282, CD284, CD286 and in a subset manner CD14, CD16, CD163, CD169CD170 and MARCO. Mouse monocytes further express F4/80 and do notexpress CD11c. Activated macrophages further express CD23, CD25, CD69and CD105.

A “dendritic cell” (DC) is an antigen presenting cell existing in vivo,in vitro, ex vivo, or in a host or subject, or which can be derived froma hematopoietic stem cell, a hematopoietic progenitor or a monocyte.Dendritic cells and their precursors can be isolated from a variety oflymphoid organs, e.g., spleen, lymph nodes, as well as from bone marrowand peripheral blood. The DC has a characteristic morphology with thinsheets (lamellipodia) extending in multiple directions away from thedendritic cell body. DCs express constitutively both MHC class I andclass II molecules, which present peptide antigens to CD8+ and CD4+ Tcells respectively. In addition, human skin and mucosal DCs also expressthe CD1 gene family, MHC class I-related molecules that presentmicrobial lipid or glycolipid antigens. The DC membrane is also rich inmolecules that allow adhesion of T cells (e.g. intercellular adhesionmolecule 1 or CD54) or that co-stimulate T-cell activation such as B7-1and B7-2 (also known as CD80 and CD86 respectively). Generally, DCsexpress CD85, CD180, CD187 CD205 CD281, CD282, CD284, CD286 and in asubset manner CD206, CD207, CD208 and CD209.

By “purified” and “isolated” it is meant, when referring to apolypeptide or a nucleotide sequence, that the indicated molecule ispresent in the substantial absence of other biological macromolecules.When referring to a cell or a population of cells, the term means thatsaid cell or said population of cells is present in the substantialabsence of other cells or population of cells. The term “purified” asused herein preferably means at least 75% by weight or number, morepreferably at least 85% by weight or number, still preferably at least95% by weight or number, and most preferably at least 98% by weight ornumber, of biological macromolecules or cells of the same type arepresent. An “isolated” nucleic acid molecule, which encodes a particularpolypeptide refers to a nucleic acid molecule which is substantiallyfree of other nucleic acid molecules that do not encode the subjectpolypeptide; however, the molecule may include some additional bases ormoieties which do not deleteriously affect the basic characteristics ofthe composition.

As used herein, the term “subject” denotes a vertebrate, preferably amammal, such as a rodent, e.g. a mouse; a feline, a canine, and aprimate. Most preferably a subject according to the invention is ahuman. Most preferably the monocytes, macrophages, or dendritic cellsexpanded according to the method of the invention are thus human cells.

In the context of the invention, the term “treating” or “treatment”, asused herein, means reversing, alleviating, inhibiting the progress of,or preventing the disease or condition to which such term applies, orone or more symptoms of such disease or condition.

Methods for Generating and Expanding Monocytes in Long Term Culture

The inventors have demonstrated that it is possible to generate,maintain and expand monocytes in culture for several months, byinactivating in said cells the expression of MafB and c-Maf.

The invention thus provides an ex vivo method for expanding monocytes,macrophages or dendritic cells, which method comprises inhibiting theexpression or the activity of MafB and c-Maf in monocytes, macrophagesor dendritic cells; and expanding the cells in the presence of at leastone cytokine or an agonist of cytokine receptor signaling.

The monocytes, macrophages or dendritic cells that serve as startingmaterial may be isolated according to any technique known in the art.

Methods for isolating starting monocytes are well known in the art andinclude those described by Fluks A J. (1981); Hardin J A. et al. (1981);Harwood R. (1974); Elias J A et al. (1985); Brandslund I et al. (1982);Pertoft H et al. (1980); Nathanson S D et al. (1977); Loos H et al.(1976), Whal S M. et al. (1984). Macrophages and dendritic cells may bederived in vitro from monocytes by differentiation (Stanley et al.,1978, 1986; Gieseler R et al. 1998, Zhou et al. 1996; Cahpuis et al1997, Brossart et al. 1998, Palucka et al 1998). In mice macrophages andDC may be obtained from spleen suspensions (Fukao, T., and Koyasu, S.,2000; Fukao, T., Matsuda, S., and Koyasu, S. 2000), from the peritonealcavity (Mishell, B. B. and Shiigi, S. M. (1980) or most commonly fromdifferent fetal liver or bone marrow progenitors using various cytokinecocktails (Ardavin et al., 2001)

One other standard method for isolating monocytes, macrophages ordendritic cells consists in collecting a population of cells from asubject and using differential antibody binding, wherein cells of one ormore certain differentiation stages are bound by antibodies todifferentiation antigens. Fluorescence activated cell sorting (FACS) maybe therefore used to separate the desired cells expressing selecteddifferentiation antigens from the population of isolated cells. Inanother embodiment, magnetic beads may be used to isolate monocytes,macrophages or dendritic cells from a cell population (MACS). Forinstance, magnetic beads labelled with monoclonal cell type specificantibodies may be used for the positive selection of human monocytes,macrophages and dendritic cells from cord blood, peripheral blood, orPBMCs, as well as pleural, peritoneal, or synovial fluids or fromvarious tissues, such as spleen and lymph node. Other methods caninclude the isolation of monocytes by depletion of non-monocytes cells(negative selection). For instance non-monocytes cells may bemagnetically labeled with a cocktail of monoclonal antibodies chosenantibodies directed against CD3, CD7, CD19, CD56, CD123 and CD235a. Kitsfor isolation of monocytes, macrophages and dendritic cells arecommercially available from Miltenyi Biotec (Auburn, Calif., USA), StemCells Technologies (Vancouver, Canada) or Dynal Bioech (Oslo, Norway).

Methods for isolation and preparation of dendritic cells and monocytesare also described in the international patent application WO2004066942and in U.S. Pat. No. 6,194,204.

As an alternative method, monocyte progenitor populations may be derivedfrom bone marrow or cord blood and differentiated to monocytes ex vivoby culture in M-CSF.

As a further alternative, dendritic cells and macrophages may be derivedfrom isolated monocytes.

For example, monocytes may be differentiated into macrophages by anytechnique well known in the art. Differentiation of monocytes tomacrophages may be induced by macrophage colony-stimulating factor(M-CSF), Recent studies have shown that optimal recombinant humanM-CSF-induced differentiation involves the autocrine activity ofsecreted interleukin 6 (IL-6), which up-regulates the expression offunctional M-CSF receptors on monocytes and enhances macrophagecytotoxicity, superoxide production, phagocytosis, chemotaxis, andsecondary cytokine secretion (Akira, 1996). The interplay between IL-6and M-CSF regulates monocyte differentiation into macrophages andinhibits DC differentiation from GM-CSF/IL-4-treated monocytes (Chomaratet al, 2000, Mitani et al., 2000).

Furthermore human monocytes may be also differentiated in vitro intomacrophages by a 7 day culture in hydrophobic bags (Chokri et al.,1989). Other techniques are also described in D'Onofrio C et al. (1983)or Gersuk G, et al. (2005). Other methods include those described bySalahuddin et al. (1982) and Hashimoto et al. (1997).

Monocytes may be differentiated into dendritic cells (DCs) by anytechnique well known in the art. For example granulocyte-macrophagecolony-stimulating factor (GM-CSF) with interleukin-4 (IL-4)differentiates monocytes into DCs. Multiple methods well known to theart have been described to differentiate human blood monocytes intodendritic cells by using GM-CSF, IL-4, and/or IFN-γ and/or CD40 ligation(Gieseler R et al. 1998, Zhou et al. 1996; Cahpuis et al 1997, Brossartet al. 1998, Palucka et al 1998). LC cells, a DC subset may be derivedusing TGF-β in addition (Strobl et al. 1997).

Methods that allow differentiation of monocyte to osteoclasts are wellknown in the art. For example M-CSF and RANKL differentiate monocytesinto OCs (Yasuda, H. et al. 1998; Hsu, H. et al. 1999).

Differentiation of the monocytes into macrophages, or dendritic cellsmay occur before inhibiting MafB and c-Maf, or after.

For instance, in a particular embodiment, the method comprises

-   -   isolating monocytes;    -   inhibiting the expression or activity of MafB and c-Maf in said        monocytes;    -   culturing the monocytes wherein the expression or activity of        MafB and c-Maf has been inhibited, in conditions allowing the        differentiation of the monocytes into macrophages or dendritic        cells.

For such differentiation, one may employ cytokines such as M-CSF.

As absence of MafB and c-Maf extends the cell expansion phase inresponse to M-CSF before its effect on macrophage differentiation,termination of the expansion phase and macrophage differentiation may beinitiated by terminating MafB and c-Maf inhibition using any of thetransient inhibition methods described below. Alternatively M-CSFconcentrations may be reduced in the medium and supplemented directlywith IL-6.

Inhibition of the expression or the activity of c-Maf and MafB may beachieved by any technique.

In a particular embodiment, the expression of MafB and c-Maf may beinhibited by using siRNA oligonucleotide, antisense oligonucleotide orribozymes

Anti-sense oligonucleotides, including anti-sense RNA molecules andanti-sense DNA molecules, would act to directly block the translation ofc-Maf and MafB mRNA by binding thereto and thus preventing proteintranslation or increasing mRNA degradation, thus decreasing the level ofc-Maf and MafB proteins, and thus activity, in a cell. For example,antisense oligonucleotides of at least about 15 bases and complementaryto unique regions of the mRNA transcript sequence encoding for c-Maf andMafB may be synthesized, e.g., by conventional phosphodiestertechniques. Methods for using antisense techniques for specificallyinhibiting gene expression of genes whose sequence is known are wellknown in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131;6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Specificmethods for preparing anti-sense oligonucleotides to c-Maf are describedin U.S. Pat. No. 6,274,338.

Small inhibitory RNAs (siRNAs) can also function as inhibitors ofexpression of c-Maf and MafB for use in the present invention. C-Maf andMafB gene expression can be reduced by contacting monocyte cell with asmall double stranded RNA (dsRNA), or a vector or construct causing theproduction of a small double stranded RNA, such that expression of c-Mafand MafB is specifically inhibited (i.e. RNA interference or RNAi).Methods for selecting an appropriate dsRNA or dsRNA-encoding vector arewell known in the art for genes whose sequence is known (e.g. seeTuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, G J.(2002); McManus, M T. et al. (2002); Brummelkamp, T R. et al. (2002);U.S. Pat. Nos. 6,573,099 and 6,506,559; and International PatentPublication Nos. WO 01/36646, WO 99/32619, and WO 01/68836). Specificmethods for preparing siRNAs against c-Maf are also described in U.S.Pat. No. 6,274,338 and for MafB in (Kim et al. 2006).

Ribozymes can also function as inhibitors of expression of c-Maf andMafB for use in the present invention. Ribozymes are enzymatic RNAmolecules capable of catalyzing the specific cleavage of RNA. Themechanism of ribozyme action involves sequence specific hybridization ofthe ribozyme molecule to complementary target RNA, followed byendonucleolytic cleavage. Engineered hairpin or hammerhead motifribozyme molecules that specifically and efficiently catalyzeendonucleolytic cleavage of c-Maf and MafB mRNA sequences are therebyuseful within the scope of the present invention. Specific ribozymecleavage sites within any potential RNA target are initially identifiedby scanning the target molecule for ribozyme cleavage sites, whichtypically include the following sequences, GUA, GuU, and GUC. Onceidentified, short RNA sequences of between about 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site can be evaluated for predicted structuralfeatures, such as secondary structure, that can render theoligonucleotide sequence unsuitable. The suitability of candidatetargets can also be evaluated by testing their accessibility tohybridization with complementary oligonucleotides, using, e.g.,ribonuclease protection assays.

Antisense oligonucleotides, siRNA oligonucleotides and ribozymes usefulas inhibitors of expression of c-Maf and MafB can be prepared by knownmethods. These include techniques for chemical synthesis such as, e.g.,by solid phase phosphoramadite chemical synthesis. Alternatively,anti-sense RNA molecules can be generated by in vitro or in vivotranscription of DNA sequences encoding the RNA molecule. Such DNAsequences can be incorporated into a wide variety of vectors thatincorporate suitable RNA polymerase promoters such as the T7 or SP6polymerase promoters. Various modifications to the oligonucleotides ofthe invention can be introduced as a means of increasing intracellularstability and half-life. Possible modifications include but are notlimited to the addition of flanking sequences of ribonucleotides ordeoxyribonucleotides to the 5′ and/or 3′ ends of the molecule, or theuse of phosphorothioate or 2′-O-methyl rather than phosphodiesteraselinkages within the oligonucleotide backbone.

Antisense oligonucleotides, siRNA oligonucleotides and ribozymes of theinvention may be delivered alone or in association with a vector. In itsbroadest sense, a “vector” is any vehicle capable of facilitating thetransfer of the antisense oligonucleotide, siRNA oligonucleotide orribozyme nucleic acid to monocytes. Preferably, the vector transportsthe nucleic acid to cells with reduced degradation relative to theextent of degradation that would result in the absence of the vector. Ingeneral, the vectors useful in the invention include, but are notlimited to, plasmids, phagemids, viruses, other vehicles derived fromviral or bacterial sources that have been manipulated by the insertionor incorporation of the antisense oligonucleotide, siRNA oligonucleotideor ribozyme nucleic acid sequences.

Methods for delivering siRNAs, ribozymes and/or antisenseoligonucleotides into monocytes are well known in the art and includebut are not limited to transfection, electroporation, microinjection,lipofection, calcium phosphate mediated transfection or infection with aviral vector containing the gene sequences, cell fusion,chromosome-mediated gene transfer, microcell-mediated gene transfer,spheroplast fusion, etc. Numerous techniques are known in the art forthe introduction of foreign genes into cells and may be used inaccordance with the present invention, provided that the necessarydevelopmental and physiological functions of the recipient cells are notdisrupted. The technique may provide for the stable transfer of the geneto the cell, so that the gene is expressible by the cell, heritable andexpressible by its cell progeny. Usually, the method of transferincludes the transfer of a selectable marker to the cells. The cells arethen placed under selection to isolate those cells that have taken upand are expressing the transferred gene. Those cells are then deliveredto a subject. A variation of the technique may provide for transienttransfer of oligonucleotides or oligonucleotide coding genes tomonocytes to enable temporary expansion of monocytes ex vivo or in vivowithout permanent genetic modification.

In a further embodiment expression of MafB and C-Maf may be inhibited bycompounds acting on promoter activity, RNA processing or proteinstability.

In another embodiment, inhibition of the activity of MafB and c-Maf maybe achieved by using mutated MafB and c-Maf polypeptides which competewith the wild-type MafB and c-Maf.

This technique is generally referred to as the technique of “dominantnegative mutants”. A dominant negative mutant is a polypeptide or anucleic acid coding region sequence which has been changed with regardto at least one position in the sequence, relative to the correspondingwild type native version at a position which changes an amino acidresidue position at an active site required for biological activity ofthe native peptide.

For example, a dominant negative mutant may consist of a truncated MafBor c-Maf molecule devoid of N-terminal effector domains that may act asa competitive inhibitor of MafB or c-Maf for DNA binding andtransactivation (Kelly et al. 2000). The efficiency of c-Maf/MafBrepression may be further improved by fusion to repressor domains thatincrease inhibitory function.

The methods of the invention are accomplished by exposing monocytes,macrophagse or dendritic cells to a dominant negative mutant in vitro.Exposure may be mediated by transfecting the cell with a polynucleotideencoding the dominant negative mutant polypeptide and expressing saiddominant negative mutant encoded by the polynucleotide so that MafBand/or c-Maf activity is inhibited. Methods for transfecting suchpolynucleotides may consist in those above described.

Exposure may also be mediated by exposing monocytes and/or macrophagseand/or dendritic cells to a dominant negative mutant polypeptidedirectly, for instance by contacting the cell with said peptidepreferably coupled to an internalization moiety. Suitableinternalization moieties are known in the art, and for instance may beselected from the group consisting of a peptide internalization sequencederived from proteins such as TAT polypeptide of HIV or Antennapedia orother homeoproteins. Alternatively transfer may be mediated by aliposome, and an antibody or an antibody fragment or ligand that bindsto a surface receptor on the target cell.

As an alternative, inhibitors of activity may consist in molecules whichare inhibitors of enzymatic posttranslational modification that regulateactivity such as phosphorylation, acetylation, methylation,ribosylation, ubiquitination, small ubiqutin like molecule modification(SUMOylation, neddylation, etc.) or molecules that alter conformation orinteraction with co-activators or co-repressors.

As a further alternative, inhibitors of activity may consist ininhibitors of DNA binding, dimerization or co-factor interaction.

Inhibitors of activity may include macromolecules or small organicmolecules. The term “small organic molecule” refers to a molecule of asize comparable to those organic molecules generally used inpharmaceuticals. The term excludes biological macromolecules (e.g.,proteins, nucleic acids, etc.). Preferred small organic molecules rangein size up to about 5000 Da, more preferably up to 2000 Da, and mostpreferably up to about 1000 Da.

The method as above described comprises a step of expanding the cells inthe presence of at least one cytokine.

Cytokines may include but are not limited to SCF, Flt3 ligand, Il-3 andM-CSF. Such cytokines are commercially available.

In a preferred embodiment, cells are maintained and expanded in thepresence of M-CSF. Macrophage colony stimulating factor (M-CSF) is amember of the family of proteins referred to as colony stimulatingfactors (CSFs). M-CSF is a secreted or a cell surface glycoproteincomprised of two subunits that are joined by a disulfide bond with atotal molecular mass varying from 40 to 90 kD (Stanley E R et al. 1997).Several secreted and membrane bound variants are known (Pixley andStanley, 2004). M-CSF is produced by macrophages, monocytes,osteoblasts, endothelial cells and human joint tissue cells, such aschondrocytes and synovial fibroblasts, in response to proteins such asinterleukin-1 or tumor necrosis factor-alpha. M-CSF stimulates theformation of macrophage colonies from pluripotent hematopoieticprogenitor stem cells (Stanley E. R., et al., Mol. Reprod. Dev., 46:4-10(1997)). M-CSF is an important regulator of the function, activation,and survival of monocytes/macrophages. Recombinant murine or human M-CSFare commercially available from R&D SYSTEMS, ABCYS, PREPOTECH, SIGMA orSTEM CELL TECHNOLOGIES.

The concentration of M-CSF in the culture medium can amount from 1 ng/mlto 100 ng/ml, preferably 5 to 50 ng/l and in a particularly preferredmanner 10 ng/l.

Alternatively it is possible to grow the cells in culture mediumcontaining 20% supernatant of L929 fibroblasts as a source of murineM-CSF (available from ATCC: CCL-1). As a source of human M-CSF theKPB-M15 cell line can be used instead.

The monocytes, macrophages or dendritic cells so obtained may becultured during a least one month, preferably at least 4, 5, 6, 7, 8, or12 months.

MafB/c-Maf Deficient Cells

The method of the invention leads to the generation of monocytes,macrophages or dendritic cells of great interest in the therapeuticfield.

An object of the invention is thus a monocyte, macrophage or dendriticcell obtainable by the method as above described. Preferably themonocyte, macrophage or dendritic cell is in isolated form.

Another object of the invention is a monocyte, macrophage or dendriticcell, which does not express MafB and c-Maf. Preferably the monocyte,macrophage or dendritic cell lacks the MafB and c-Maf genes.

The monocyte, macrophage, or dendritic cell may be of any species. It ispreferably from a murine origin, or from a human origin.

When the cell is a dendritic cell, it may be useful to sensitize thecell to antigens. For that purpose, one may contact the dendritic cellswith the antigenic molecule of interest or antigenic peptides for about30 minutes to about 5 hours (“peptide or antigen pulsing”). One can alsocontact dendritic cells with cells or membranes of cells expressingantigens or antigenic peptides, with liposomes containing antigens orantigenic peptides or with RNAs coding for antigens or antigenic.

A particular subject of the invention is thus a dendritic cell asdefined above, i.e. MafB and c-Maf deficient, which is further loadedwith an antigenic molecule.

The antigenic molecule may be any molecule against which an immuneresponse is sought. Examples of antigen molecules comprise for instanceviral proteins or peptides, bacterial proteins or peptides, or tumorantigens, such as MART-1, MAGE, BAGE, PSA, p53, Rb, Ras, etc.

Mouse Monocytes

Another object of the invention relates to a method for generatingmurine monocytes wherein said method comprises the steps consisting of:

-   -   i) isolating monocytes derived from a mouse deficient for MafB        and c-Maf factors and    -   ii) culturing said cells in the presence of M-CSF

Mouse embryos deficient for MafB and c-Maf may be obtained through thecrossing of MafB deficient mice with c-Maf deficient mice. Generation ofMafB deficient mice has been previously described (Blanchi B. et al.,2003). The generation of c-Maf deficient mice has also been described(Kim J L. et al. 1999). Mice with a MafB and c-Maf deficienthematopoietic system may be obtained by reconstituting irradiated micewith MafB and c-Maf deficient fetal liver cells. Such a method isdescribed in the below example.

Another object of the invention relates to a MafB/Cmaf deficient murinemonocyte obtainable by tissue specific deletion of MafB and c-Maf usinga loxP/Cre recombinase system.

Another object of the invention relates to a MafB/c-Maf deficient murinemonocyte obtainable by the method as above described.

MafB/c-Maf deficient murine macrophages, or dendritic cells may beobtained through the differentiation techniques as above described.

Cell Therapy:

According to the present invention, monocytes, macrophages anddendritics cells can be easily and effectively generated in vitro. Theability to obtain a large number of in vitro expanded monocytes,macrophages and dendritic cells opens new opportunities for thetherapeutic field.

The invention thus provides a pharmaceutical composition comprising amonocyte, macrophage or dendritic cell as defined above, in combinationwith a pharmaceutically acceptable carrier.

The invention further provides a pharmaceutical composition whichcomprises the dendritic cell as defined above, loaded with an antigenicmolecule, for use as a vaccine.

The mononuclear phagocyte system (monocyte and macrophages) represents adistributed organ responsible for homeostasis within the host. Saidsystem is involved in every disease process in which there is persistenttissue injury or metabolic disturbance. Macrophages and monocytesmediate acute as well as chronic inflammation, and promote repairthrough removal of dead cells and fibrin by phagocytosis andfibrinolysis, induce blood vessel ingrowth (angiogenesis) and modulatefibroblast invasion and production of extracellular matrix. They producemediators that mobilize systemic responses of the host including fever,release and catabolize stress and other hormones, increase metabolicactivity of other cells, and influence blood flow to tissues andcapillary permeability. The macrophages themselves display considerableheterogeneity in their functions, often expressing activators as well asinhibitors of a property, e.g. proteolytic activity, or pro- andanti-inflammatory cytokine production, depending on the evolution of aparticular host response.

It is therefore described a method for treating a subject affected witha disease resulting from a deficiency in the monocyte compartment, whichmethod comprises administering said subject with monocytes, macrophagesor dendritic cells, in which MafB and c-Maf expression or activity isinhibited, preferably with MafB and c-Maf deficient monocytes,macrophages or dendritic cells.

A further object of the invention is the use of a monocyte, macrophageor dendritic cell as defined above for the manufacture of a medicamentintended for the treatment of a disease selected from the groupconsisting of a cancer, acute or acquired immuno-deficiencies, chronicor acute injury, wounds, degenerative diseases, autoimmune diseases,chronic inflammatory diseases, atherosclerosis, poly- andosteo-artritis, osteoporosis, infectious diseases (e.g. infections byvirus, or bacteria), and metabolic diseases.

Immunodeficiencies include acquired or genetic in origin, or as a resultof radiotherapy/chemotherapy. AIDS is particularly contemplated. Also,the invention offers the possibility for the development ofantigen-specific cancer immunotherapies.

Macrophages according to the invention may be useful for the treatmentof HIV infections. Dysfunction of neutrophils (polymorphonuclearleukocytes [PMNL]) and macrophagic cells occurs indeed as a consequenceof human immunodeficiency virus type 1 (HIV-1) infection. Macrophagescontribute to the resolution of early inflammation ingesting PMNLapoptotic bodies. A recent study suggests that impaired macrophagephagocytosis of PMNL apoptotic bodies may contribute to the persistenceof the inflammatory state in HIV-infected subjects, especially duringopportunistic infections that are often favored by defective phagocyticactivity (Torre D et al. 2002). Therefore, methods for generatingmacrophages as above described may be useful for the treatment ofsubjects infected with HIV.

Patients on chemotherapy with anticancer agents such as cyclophosphamide(CP) experience a strong reduction in the size of tissue macrophagepopulations that accompanies blood leukopenia. These patients are thusespecially susceptible to opportunistic infections, includinggram-negative bacterial pneumonia. Such opportunistic infections are acommon cause of death in cancer patients who are undergoing chemotherapy(Santosuosso M, et al. 2002). Therefore, methods for generatingmacrophages as above described may be useful for the treatment ofsubjects who have undergone a chemotherapy.

Macrophages may inhibit precursor cells apoptosis in a cell to cellcontact and may serve as stromal support for efficient cellularengraftment for tissue repair (see for example document WO2005014016).In particular macrophages could inhibit myogenic precursor cellsapoptosis. Therefore methods for generating macrophages as abovedescribed may be useful for the treatment of lesions such as bone ormuscular lesion, possibly resulting from a disease or an injury. It canbe for example a bone fracture, or a torn muscle. In a more particularembodiment of the invention, said lesion is a cardiac lesion or injury.In particular, it can be for example myocardial infarction, heartinsufficiency, coronary thrombosis, dilated cardiomyopathy or anycardiomyocyte dysfunction subsequent to, or resulting from, any geneticdefect. Therefore, methods for generating macrophages as above describedmay be useful for the treatment of acute cardiac insufficiencies withbad prognostic despite progress in treatments, such as infiltrativecardiomyopathy, or cardiomyopathy due to anthracyclin toxicity orcardiomyopathy secondary to HIV infection.

Methods for generating macrophages as above described may be also usefulfor the treatment of spinal cord injury. A therapy for complete spinalcord injury (SCI) may be indeed consisting in autologous grafts ofmacrophages that have been educated to a wound-healing phenotype byco-incubation with skin tissue (Schwartz M et al. 2006).

Macrophages are essential for wound healing and thus methods forgenerating macrophages as above described may be useful for enhancingwound healing and/or repairing tissue damage. The wound healing processhas indeed 3 phases. During the inflammatory phase, numerous enzymes andcytokines are secreted by the macrophage. These include collagenases,which clear the wound of debris, interleukins and tumor necrosis factor(TNF), which stimulate fibroblasts (to produce collagen) and promoteangiogenesis; and transforming growth factor α (TGFα), which in healingskin wounds stimulates keratinocytes. This step marks the transitioninto the process of tissue reconstruction, ie, the proliferative phase.

Macrophages play a major role in chronic inflammatory and auto-immunedisease and usually are activated or hyperactivated in a specific wayunder these conditions. Monocytes may be amplified by the describedmethod to be genetically modified, pharmacologically treated oralternatively activated or stimulated by bio-active molecules such ascytokines or growth factors to present a desirable phenotype beforeintroduction into a subject to compete with and supplant macrophageswith undesired activity.

Cancer is the second leading cause of death in most developed countriesaccounting for about 150,000 and 550,000 deaths each year in France andUSA, respectively. Despite the fact that more than half of all cancercases can be cured, the average cancer patient, not curable by surgeryor radiotherapy, still has a less than 10% chance of being cured by anyother treatment (A. Grillo-Lopez, 2003). It has long been hypothesizedand it is now recognized that the immune system plays a role in cancersurveillance and in the prevention of tumors.

Macrophages are a major component of the leukocyte infiltrate of tumorsand have been shown to have both positive and negative effects on tumorformation and progression, where the final outcome depends to a largedegree on micro-environment dependent polarization of tumor associatedmacrophages (TAM). Ex vivo amplified and modified macrophages couldtherefore be therapeutically useful to bring macrophages of desiredpolarization profile or that carry therapeutic gene constructs orreagents to the tumor site (Bingle et al., 2002) (Mantovani et al.,2002). Moreover dendritic cells (DC) prime naive T cells to becomeeffector cells able to provide long-term protection against tumorrecurrence. Cell therapy based on these two types of cells appearstherefore as a promising tool to treat cancer patients. Thereforemethods for generating macrophages and/or dendritic cells as abovedescribed may be useful for treating cancer diseases.

Dendritic cells may be induced to mature (maturing DC) through a shorttreatment with a bacterial extract and interferon-gamma. Furthermore,therapeutic vaccination against tumors has been shown to providelong-term protection in animal models. Tumor (or tumor cell line)lysates constitute an attractive source of multiple tumor antigens totrigger both CD8 and CD4 T cell responses. Therefore pulsing dendriticcells with such tumor antigens represents a tool for the treatment ofcancer.

Therefore methods of the invention may be useful for preparingpharmaceutical compositions comprising macrophages and/or dendriticcells for treating cancer. Cancer include carcinomas of breast, colon,rectum, lung, oropharynx, hypopharynx, esophagus, stomach, pancreas,liver, gallbladder and bile ducts, small intestine, kidney, bladder,urothelium, female genital tract, (including cervix, uterus, and ovariesas well as choriocarcinoma and gestational trophoblastic disease), malegenital tract (including prostate, seminal vesicles, testes and germcell tumors), endocrine glands (including the thyroid, adrenal, andpituitary glands), and skin, as well as hemangiomas, melanomas, sarcomas(including those arising from bone and soft tissues as well as Kaposi'ssarcoma) and tumors of the brain, nerves, eyes, such as astrocytomas,gliomas, glioblastomas, retinoblastomas, neuromas, neuroblastomas,Schwannomas, and meningiomas, and tumors arising from hematopoieticmalignancies such as leukemias as well both Hodgkin's and non-Hodgkin'slymphomas.

In treating cancer, for example, dendritic cells can be pulsed withtumor antigens and administered to a patient to treat, e.g., establishedtumors, or to prevent tumor formation, as discussed above.

In another embodiment a dendritic cell of the invention may be fused toa cancer cell and therefore can be administered to the patient, whereinthe fused dendritic cell will, in its role as an antigen-presentingcell, present the antigen to the immune system. Dendritic cells can befused with other cells, e.g., cancer cells, by any method known in theart. For example, methods for fusing dendritic cells with cancer cells,as well as methods for administering them to animals have been describedin Gong et al. (Nat. Med. 3 (5): 558-561 (1997)) and Guo et al. (Science263: 518-520 (1994)). The cancer cell can be any type of cancer cell tobe targeted in a patient, e.g., cancer cells of the breast, liver, skin,mouth, pancreas, prostate, urinary tract, e.g., bladder, uterus, ovary,brain, lymph nodes, respiratory tract, e.g., larynx, esophagus, andlung, gastrointestinal tract, e.g., stomach, large and small intestine,colon, or rectum, bone, blood, thyroid, and testes, or any cancer cellline known in the art to be suitable for fusing to other cells e.g.,dendritic cells.

Similar vaccination protocols may be applied to infectious diseases byloading the dendritic cells with pathogen (viral, bacterial or parasite)antigen.

In a preferred embodiment, monocyes, macrophages, or dendritic cells areobtained directly from the subject to whom they are administered. Inthat case the transplantation is autologous. But in another embodimentthe transplantation can also be non-autologous. For non-autologoustransplantation, the recipient is preferably given an immunosuppressivedrug to reduce the risk of rejection of the transplanted cell. Methodsof administering cells according to the invention include but are notlimited to intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal, and epidural routes. The cells may beadministered by any convenient route, and may be administered togetherwith other biologically active agents. The route of administration ispreferably intravenous or intradermal. The titer of monocytes and/ormacrophages and/or dendritic cells transplanted which will be effectivein the treatment of a particular disease or condition will depend on thenature of the disorder or condition, and can be determined by standardclinical techniques. In addition, in vitro assays may optionally beemployed to help identify optimal dosage ranges. The precise dose to beemployed in the formulation will also depend on the route ofadministration, and the seriousness of the disease or disorder, andshould be decided according to the judgment of the practitioner and eachsubject's circumstances.

Pharmaceutical Compositions

The present invention provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of a monocyteand/or macrophage and/or dendritic cell produced according to theinvention, and a pharmaceutically acceptable carrier or excipient. By a“therapeutically effective amount” of a cell as above described is meanta sufficient amount of said cell to treat a disease or disorder at areasonable benefit/risk ratio applicable to any medical treatment. Itwill be understood, however, that the total daily usage of compositionsof the present invention will be decided by the attending physicianwithin the scope of sound medical judgment. The specific therapeuticallyeffective dose level for any particular patient will depend upon avariety of factors including the disorder being treated and the severityof the disorder; activity of the specific compound employed; thespecific composition employed, the age, body weight, general health, sexand diet of the patient; the time of administration, route ofadministration, and rate of excretion of the specific compound employed;the duration of the treatment; drugs used in combination or coincidentalwith the specific cells employed; and like factors well known in themedical arts

Pharmaceutically acceptable carrier or excipient includes but is notlimited to saline, buffered saline, dextrose, water, glycerol andcombinations thereof. The carrier and composition can be sterile. Theformulation should suit the mode of administration. The composition, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. The composition can be a liquidsolution, suspension, or emulsion. In a preferred embodiment, thecomposition is formulated in accordance with routine procedures as apharmaceutical composition adapted for intravenous administration tohuman beings. Typically, compositions for intravenous administration aresolutions in sterile isotonic aqueous buffer. Where necessary, thecomposition may also include a solubilizing agent and a local anestheticsuch as lignocaine to ease pain at the site of the injection.

Methods for Engineering Cells of the Invention

Monocytes, macrophages and dendritic cells of the invention may befurther genetically engineered so that said cells express a therapeuticnucleic acid of interest, which encodes a protein of interest.

Suitable gene of interest include growth factors. For instance, cells ofthe invention can be genetically engineered to produce gene productsbeneficial upon transplantation of the genetically engineered cells to asubject. Such gene products include, but are not limited to,anti-inflammatory factors, e.g., anti-TNF, anti-IL-1, anti-Il-6,anti-IL-2 . . . etc. Alternatively, cells of the invention can begenetically engineered to “knock out” the expression of MHC in order tolower the risk of rejection.

Macrophages have been shown to fuse with muscle cells or hepatocytes andcan correct a genetic defect in these cells (Camargo et al., 2003)(Camargo et al., 2004) (Willenbring et al., 2004). Cells of theinvention may therefore be also engineered to express multiple or singlecopies of normal or hyperactive variants of genes that are mutated ingenetic disorders. Examples include but are not limited to enzymedeficiencies in the liver or dystrophin in Duchenne muscular dystrophy.

Macrophages are a major component of the tumor infiltrate. Suitablegenes of interest to be expressed by the cells of the invention maytherefore also be genes that carry anti-tumor activity.

Furthermore cells of the invention can be engineered to inhibitexpression of genes by siRNA or antisense or siRNA or antisense encodinggenes that are stably or transiently introduced into the cells. Targetsfor inhibition include but are not limited to inflammatory cytokines,proteases, transcription factors and enzymes affecting inflammatorypathways.

In addition, cells of he invention can be genetically engineered forexpressing a growth factor that promotes differentiation and/orproliferation.

Monocytes, macrophages and dendritic cells of the invention may befurther engineered so that said cells carry a molecule of interest.

Suitable molecule (or even genes) of interest include proteaseinhibitors or knockdown of proteases, transcription factors or dominantversions thereof to globally inhibit expression of inflammatorymediators, cytokines, chemokines, proteases or to globally induceanti-inflammatory mediators, cytokines, chemokines and proteaseinhibitors. Genes of interest may encode for cytokines and enzymes thatselectively effect M1/M2 polarization of macrophages (Il-4, Il-10,Il-13, TGFβ), or for cytokines inhibiting osteoclast differentiation(such as anti RANKL, OPN), or for growth factors and protease inhibitorsstimulating wound healing (such as PDGF, EGF, SLPI), or foranti-microbial peptides.

The delivery of said genes may be performed by any method well known inthe art as above described.

Regenerative Medicine

Because of the inadequate supply of donor organs, alternatives toallografts are desperately needed. Cell-replacement therapies mayprovide a promising alternative to liver, pancreas, or islet celltransplantation. Such therapies may also provide treatment options inorgan systems where transplantation is not possible or indicated. It hasbeen recently shown that not only stem or progenitor cells but alsocommitted myelo-monocytic cells, including mature monocytes, macrophagesprovide a significant potential for targeted and well-tolerated celltherapy aimed at organ regeneration, especially in the liver andpancreas. It has been shown that such mature myelo-monocytic cells ofboth murine and human origin can significantly contribute to liver andbeta-islet pancreatic tissue in different mouse models and performtissue specific functions in these organs (Camargo, F. D., Green, R., etal, 2003; Willenbring, Bailey et al. 2004; Ruhnke, Ungefroren et al.2005). Although the mechanism of this is not entirely clear, theseobservations indicate that the administration of macrophages directly tothe damaged organ or systemic transplantation of their proliferativeprogenitors represents an attractive and little invasive therapeuticstrategy. The prospect of a clinical application of this approach,however, is hampered by the difficulty to amplify sufficient numbers ofmonocytes in culture.

The monocytes, macrophages or dendritic cells obtained by the method ofthe invention can be used in regenerative medicine, e.g. foradministration directly to a damaged organ, or systemic transplantation.

Screening Methods

In certain diseases, it may be desired either to destroy or reduce thenumber of monocytes, macrophages or dendritic cells, or to target andtreat infected monocytes, macrophages or dendritic cells. In such cases,it is desired to develop drugs that target monocytes, macrophages, ordendritic cells.

The method of the invention for generating monocytes, macrophages, ordendritic cells may be useful for screening such drugs.

A general method for screening drugs, which method comprises contactinga monocyte, a macrophage, or dendritic cell as defined above, with acandidate compound, and determining the ability of said compound tobind, and optionally destroy, said cell, to inhibit its replication, orto modify its behaviour. In particular, the candidate compound maychange the behaviour of the cell (e.g. its ability to differentiate) inresponse to a particular stimulus, e.g. a cytokine.

For example, several pathogens are able to subvert the phagocytoseprocess by a range of stratagems utilizing a vacuolar pathway forinvasion and survival, even in macrophages. Other examples are known bywhich organisms evade ingestion (mycoplasma), destroy opsoninsenzymatically, inhibit fusion and acidification (mycobacteria) andrecruit novel membranes (Legionella) as well as other organelles.Trypanosoma cruzi and Candida albicans rapidly recruit lysosomes,perhaps to promote their own differentiation. Leishmania multipliesfreely within phagolysosomes whereas Listeria monocytogenes disruptslysosomal membranes and escapes into the cytoplasm, where it initiatesactin polymerization for intracellular movement and intercellularspread. Bacteria of the genus Brucella are intracellular pathogenscapable of survival and replication within macrophages of mammalianhosts. This pathogen uses multiple strategies to circumvent macrophagedefence mechanisms and generate an organelle permissive for replication.Finally, the facultative intracellular pathogen Salmonella entericatriggers programmed cell death in macrophages.

Therefore the methods for generating macrophages as above described maybe useful for screening drugs against pathogens such as Mycoplasma,Mycobacteria, Legionella, Trypanosoma, Leishmanias, Listeria, Brucellaor Salmonella.

The macrophage also contributes to the initial infection, disseminationand persistence of human immunodeficiency virus type 1 (HIV-1) in thebody. Known factors that influence infection of macrophages by differentHIV strains include CD4 and chemokine coreceptors for viral entry.

Therefore the methods for generating macrophages as above described maybe useful for screening drugs against HIV infections.

Methods of the invention may be also useful for screening drugs thatinhibit an inflammatory response in macrophages for use against chronicinflammatory and autoimmune disease (such as polyarthritis, Crohnsdisease or multiple sclerosis) or cancer with a strong contribution ofinflammatory tumor associated macrophages.

Methods for generating macrophages as above described may also be usefulfor screening drugs promoting wound healing or reducing scarring.

Methods for generating macrophages as above described may be useful forscreening drugs inhibiting osteoclast differentiation for use inosteoarthritis and osteoporosis.

FIGURES

FIG. 1: 20.000 fetal liver cells from wt (open bars) or MafB/c-Mafdouble deficient (filled bars) E14.5 embryos were incubated in semisolidmedium containing a cytokine mix of recombinant SCF, GM-CSF, IL-3, IL-6,and EPO (A) or 10 ng/ml of recombinant M-CSF (B), GM-CSF (C), G-CSF (D)or Il-3 (E) only. Colonies were scored after 12 days and the averagenumbers of CFU-M, CFU-E, CFU-GEMM, CFU-GM and CFU-G are indicated.Experiments were performed in duplicate and error bars indicate standarderror of the mean from three individual embryos of each genotype.

FIG. 2: 10.000 fetal liver cells from wt (diamonds) or MafB/c-Maf doubledeficient (squares) E14.5 embryos were incubated in semisolid mediumcontaining 10 ng/ml of recombinant M-CSF, GM-CSF, or Il-3 as indicated.Colonies were scored every 8 days, washed out from the medium andreplated at the same concentration and under the same conditions. Assayswere performed in duplicate and two independent experiments showedequivalent results.

FIG. 3: Fetal liver cells from wt, MafB deficient, c-Maf deficient orMafB/c-Maf double deficient E14.5 embryos as indicated weredifferentiated to monocytes/macrophages for 9 days in L-cell conditionedmedium as a source of M-CSF, simulated for 24 h with the indicatedconcentration of recombinant M-CSF and assayed for proliferation bymonitoring cells in S-phase after 18 h of BrdU incorporation. Theproliferation index as the ratio of proliferation in the stimulated tothe non-stimulated cultures is indicated. Error bars indicate standarderror of the mean from triplicate samples and two independentexperiments showed equivalent results.

FIG. 4: Three independent monocyte cultures were derived from M-CSFcolony assays of MafB/c-Maf double deficient E14.5 fetal liver cells bywashing out the cells from the semi-solid medium and incubating them inL-cell conditioned medium as a source of M-CSF. Medium was changed every4 days and cultures were counted and passaged at confluency. The graphshows the calculated total number of cells derived after the indicatednumber of passages.

FIG. 5 Phenotypic and functional characterization of MafB/c-Maf doubledeficient monocyte cultures: A. Giemsa staining of a cytospin fromMafB/c-Maf double deficient monocyte cultures showing a homogenousmonocyte/macrophage morphology. B. Phase contrast photomicrograph ofMafB/c-Maf double deficient monocyte cultures C./D. FACS profiles ofMafB/c-Maf double deficient monocyte cultures stained for antigens ofthe myeloid lineage (C) or other lineages (D). Cells were nearly 100%positive for monocyte/macrophage antigens (Mac-1, F4/80 and FcgRII/III)but negative for the granulocytic marker Gr-1, the progenitor markersc-kit and CD34, the erythroid marker Ter119, and the T-lymphoid markersCD3 and CD4. They were also negative for the B-lymphoid marker CD19 (notshown). E. MafB/c-Maf double deficient monocyte cultures (DKO cells)were incubated with PE-coated latex beads and analyzed by FACS foringested fluorescent beads as a measure of phagocytosis. For comparisonphagocytic activity of the RAW 267 macrophage cell line is shown as apositive control. The negative control shows cells not incubated withbeads.

FIG. 6: Derivation of monocyte cultures from MafB/c-Maf deficient bloodof aged mice. A.-D. 100.000 white blood cells (WBC) form peripheralblood of 22 months old mice reconstituted either with wt or MafB/c-Mafdouble deficient fetal liver cells at 2 months of age were incubated insemisolid medium containing 100 ng/ml recombinant M-CSF and analysed forcolony formation after 12 days. Photomicrographs of colonies in wt (A)or MafB/c-Maf deficient (B) samples and graph (C) showing the totalnumber of colonies from wt or MafB/c-Maf deficient samples. Error barsindicate standard error of the mean of duplicate samples from twoindividual mice of each genotype. A magnification of an individualcolony in B is shown in panel D. E., F. 100.000 WBC form peripheralblood of wt or MafB/c-Maf deficient reconstituted mice were cultured inL-cell conditioned medium as a source of M-CSF, counted and passagedevery 8 days. A phase contrast photomicrograph of a MafB/c-Maf deficientmonocytic culture is shown in E and the calculated total number of cellsat each indicated passage number is shown in F.

EXAMPLE

Material and Methods

Mice: We described previously the generation of MafB deficient mice on a129Sv-C57BL/6 background and their genotyping by PCR with primers formafB and gfp, replacing mafB in the knockout allele (Blanchi et al.2003). The generation of c-Maf deficient mice has also been described(Kim et al. 1999). MafB+/− and c-Maf+/− mice were crossed to obtainMafB; c-Maf+/−; +/− mice, which were intercrossed to obtain MafB;c-Maf−/−; −/− embryos. All experiments were performed in accordance withinstitutional guidelines using mice maintained under specificpathogen-free conditions.

Fetal Liver cell preparation: Embryos were collected aseptically atembryonic day E14.5. A single cell suspension was prepared in primarycell medium (IMDM, 20% FCS, 1% penicillin/streptomycin) from the liverof each embryo and stored at 4° C. until the genotype was confirmed byPCR.

Colony assays: 2×10⁴ fetal liver cells were seeded in semi-solid mediumcontaining MethoCult M3232 containing a mix of cytokines (SCF, Epo,GM-CSF, Il-3, Il-6) or MethoCult M3234 (Stem Cell Technologies,Vancouver, Canada) supplemented with 10 ng/ml murine rM-CSF, 10 ng/mlmurine rGM-CSF, 10 ng/ml murine rG-CSF or 10 ng/ml murine rIl-3 (PeproTech Incorporation) according to manufacturer's instructions. Briefly,300 ml of cell containing medium was mixed with 300 ml cytokinecontaining medium, added to 1400 ml of methylcellulose medium and mixedvigorously before plating 900 ml each of this mix in duplicate 35 mmplates. Size and total number of colonies with more than 16 cells werescored in duplicate plates after 4, 6, 9 and 12 days.

Replating assays: 1×10⁴ fetal liver cells per plate were seeded insemi-solid medium containing methyl cellulose (M3234, Stem CellsTechnologies) supplanted with 50 ng/ml murine rM-CSF, murine rGM-CSF ormurine rIl-3 (allPepro Tech Incorporation). After 8 days colonies werecounted and cells were washed repeatedly in medium to removemethyl-cellulose until a single cell suspension was obtained. 1×10⁴cells were re-plated under the same conditions and colony formation wasscored again after day 8. Re-plating was repeated three times.

Growth of MafB/c-Maf deficient cells in liquid culture: After the 4thplating only MafB/c-Maf deficient cells under M-CSF conditions stillgave colonies. These were repeatedly washed in medium to removemethyl-cellulose and taken into liquid culture at 1×10⁴/100 ml DMEM/10%heat inactivated FCS/1% penicillin/streptomycin and 1% Na-Pyruvate,supplemented with 20% L-cell supernatant as a source of M-CSF. Culturein recombinant murine M-CSF resulted in identical growthcharacteristics. Cells were subjected to a partial medium change every 4days and split 1:4 with a complete medium change every 8 days.

L-Cell supernatant production: L929 fibroblasts (available from ATCC:CCL-1) were used as source of murine M-CSF conditioned medium. L-Cellswere cultured and maintained in L-cells growth medium (DMEM, 10% FCS HI,1% Na Pyrute, 1% penicillin/streptomycin). For producing supernatant,cells were grown to 70% confluency and medium was changed to L-cellssupernatant medium (IMDM, 2% FCS HI, 1% Na Pyruvate, and 1%Penicillin/Streptomycin). Supernatant was collected after 5 days andfurther medium was added for another 5 days. After collecting the secondsupernatant both were pooled and filtered through 0.22 mm filter andstored in aliquots at −20° C.

FACS Analysis: For antibody staining cells were resuspended in filteredFACS medium (02% FCS and if necessary FcgII/III blocking antibody inPBS) at a concentration of 1×10⁶ to 1×10⁷ cells/ml followed byincubation at 4° C. for 30 min with properly diluted, fluorochromemonoclonal antibodies. Directly flurochrome labelled antibodies againstmouse antigens were purchased from BD or from eBiosciences. Afterwashing twice with PBS cells were analysed on a FACSCalibur or FACSCanto machine (Becton-Dickinson, San Jose, Calif.). Data were andanalysed with Cell Quest® (Becton-Dickinson) or Flowjo® software.

Phagocytosis assay: Fluorescent beads (Molecular Probes, 1 μM, F-8851)were washed once in sterile PBS, resuspended in DMEM/10% FCS andsonicated for 10 intervals at 10 seconds each. 25 μl of bead solutionwas incubated macrophages in 24-well plates for 2 hours. Cells were thenextensively washed with PBS, fixed with 1% PFA and analyzed by flowcytometry on a FACSCalibur (Beckton Dickinson).

Bone marrow reconstituted mice: For reconstitution experiments a singlecell suspension from fetal liver cells of the same genotypes (MafB;c-Maf−/−; −/− or WT) were pooled, filtered through 100 μm gaze, washedonce with either 1×HBSS or PBS and re-suspended in PBS or HBSS forinjection. 1×10⁶ FL cells in 200 μl of PBS/HBSS were injected into thetail vein of lethally irradiated (900-1000 rad) age- and sex-matchedLy5.1 recipient mice. Irradiation was done at least 4 hours before celltransfer and mice were kept on antibiotics in the drinking water for 4weeks post-transplantation.

Proliferation assay: Fetal liver cells were differentiated for 9 days inM-CSF containing medium, washed in PBS and plated in a 96 wellflat-bottom plate at 20,000 cells/well in 200 μl of medium containingthe indicated concentrations of recombinant M-CSF. After 36 h BrdU wasadded at a final concentration of 10 nM and 18 hours later cells werefixed and labeled with anti BrdU-antibody following the protocol of acell proliferation ELISA BrdU calorimetric kit (Roche cat #1 647 229).Briefly, wells were washed 5× with washing solution and incubated with100 μl detection reagent. After 20 minutes the ELISA reaction wasstopped by adding 25 μl of 1M H2SO4 and the colour product was measuredin a spectrophotometer at 450 nm.

Results

MafB, c-Maf double deficient fetal liver cells have increased monocyticcolony forming potential: Since MafB deficiency is neonatally lethal, weanalyzed the effect of MafB/c-Maf double deficiency on hematopoiesis inthe E14.5 fetal liver. FACS staining for lineage specific surfacemarkers revealed no abnormalities of lineage distribution in MafB/c-Mafdeficient E14, 5 fetal liver. To quantify the number of cells that couldproliferative in response to specific cytokines, MafB/c-Maf deficient orwild type (WT) control cells from E14,5 embryos were cultured in methylcellulose medium either with a mix of cytokines (Il-3, Il-6, GM-CSF, SCFand Epo; FIG. 1A) or individual myeloid cytokines (M-CSF, GM-CSF, G-CSFor Il-3, FIG. 1B-E) o. Monocytic colonies (CFU-M) were significantlyincreased for MafB/c-Maf deficient compared to WT foetal liver cellsunder all conditions that support monocytic growth, (M-CSF, GM-CSF, IL-3and mixed conditions), whereas other types of colonies remained in thenormal range. Furthermore such an increase in monocytic colonies was notseen in individual MafB or c-Maf deficient foetal liver cells (notshown). This indicated that the combined loss of MafB and c-Maf resultedin an increased number of cells giving rise to monocytic colonies inresponse to myeloid cytokines.

MafB, c-Maf double deficient CFU-M monocytic cells have enhanced selfrenewal capacity in M-CSF: To further analyze, whether MafB/c-Mafdeficient monocytic cells maintained self renewal potential, we testedthe serial replating ability of cells from MafB/c-Maf deficient CFU-Mcolonies. In such assays colonies were washed out from methocell,dissociated and replated under the same cytokine conditions in freshmethocell cultures.

As shown in FIG. 2 under GM-CSF and Il-3 conditions colonies could bereplated both from wt and MafB/c-Maf deficient assays for 3 times but nocolonies formed after the 4^(th) re-plating. By contrast dramaticdifferences between WT and MafB/c-Maf KO cells were observed under M-CSFconditions. Whereas WT colonies already disappeared after the secondre-plating, MafB/c-Maf deficient cells were forming continuouslyincreasing numbers of colonies in M-CSF up to the 4^(th) re-plating.This indicated that MafB/c-Maf deficient foetal liver derived monocyticcells had the ability to specifically self renew and expand in M-CSF.

MafB/c-Maf deficient monocytes show increased proliferation in responseto M-CSF: To test whether this increased self renewal potential was dueto increased M-CSF dependent proliferation of MafB/c-Maf deficientmonocytes we differentiated fetal liver cells of wt, MafB deficient,c-Maf deficient or MafB/c-Maf deficient E14.5 embryos into monocytes for9 days and then stimulated these cells with different concentrations ofM-CSF for 36 hours. We monitored their rate of DNA synthesis in responseto M-CSF and established their proliferation index by comparing the rateof BrdU incorporation after 18 h in stimulated and unstimulated cells.As shown in FIG. 3 wt, MafB deficient or c-Maf deficient monocytesshowed nearly no proliferative response to M-CSF. By contrast MafB/c-Mafdeficient monocytes showed a dramatic increase of proliferation inresponse to M-CSF. This indicated that the observed continued expansionMafB/c-Maf deficient monocytes is due to a strongly increased ability toproliferate in response to M-CSF.

MafB/c-Maf deficient monocytes can be maintained in M-CSF culture forseveral months and expanded by more than 10¹⁰ fold: To further test howlong this enhanced self renewal and M-CSF dependent proliferationcapacity of MafB/c-Maf deficient monocytes could be maintained, CFU-Mmonocytic colonies were taken into liquid culture and cultured in thepresence of M-CSF containing medium. Under these conditions the cellscontinued to proliferate and expand in culture. Four cell populationswere independently derived from different embryos and replating assaysand could be maintained in M-CSF containing culture for at least 15passages or 4 months without any sign of slowed growth or crisis. Twopopulations went into crisis at passage 16 but the other two did notshow any sign of crisis up to passage 24 or 6.5 months and one of themhas been kept continuously in culture for 18 months without crisis orslowed growth. The growth curves up to passage 15 of three of thesepopulations (named DKO 42, 46 and 56) are shown in FIG. 4 Cells frozenat each passage could be easily taken into culture again. The derivedpopulations could also be cloned and at least 6 independent linesderived from individual cells of early passage populations have beenestablished.

The total number of cells obtainable without crisis in long term culturewas calculated to represent an amplification factor of at least 10¹⁰. Toillustrate this enormous amplification factor, the monocytes present ina typically volume of a routine blood analysis (about 5 ml) wouldtheoretically be sufficient to generate the total monocytes of 10Million people, if similarly amplified.

c-Maf/MafB deficient monocytic cells in M-CSF cultures have a normalmonocyte/macrophage phenotype: c-Maf/MafB deficient cells that werecontinuously cultured in the presence of M-CSF for extended periods oftime maintained a monocyte/macrophage phenotype. As shown in FIG. 5Ac-Maf/MafB deficient cells showed a typical macrophage morphology inGiemsa/May-Gruenwald staining. Their appearance in culture under phasecontrast was less flattened than typical macrophage cultures, which islikely an indication of their continued proliferation (FIG. 5A). FACSanalysis also showed that the total population ofc-Maf/MafB deficientcells expressed the typical monocyte/macrophage markers F4/80, Mac-1(CD11b), M-CSFR (CD115) and FcgRII/III (CD16/CD32) but none of selectedT-Cell (CD3, CD4, CD8), B-cell (CD19), erythroid, (Ter-119),granulocytic (Gr-1) markers (FIGS. 5C,D). In cultures containing lowamounts of M-CSF but not high amounts of M-CSF, CD11c was observed to beupregulated, suggesting that these cells have the potential for DCdifferentiation, which can be suppressed by the continuous presence ofM-CSF. Furthermore c-Maf/MafB deficient cells exhibited typicalmonocyte/macrophage function, as they were able to quantitativelyphagocytose large amounts fluorescent latex beads to a degree equal orhigher than wt primary macrophages or macrophage cell lines (FIG. 5E).

Together these observations indicated that despite their continuedproliferation in culture all cells maintained a mature phenotype offully differentiated and functional monocytes/macrophages.

c-Maf/MafB deficient monocytes and macrophages do not developmalignancies in vivo: Increased replating efficiency in vitro is alsoobserved for malignant transformed cells that have leucemic potential invivo. To control for this possibility we reconstituted lethallyirradiated recipient mice with c-Maf/MafB deficient fetal liver cellsand observed the reconstituted mice for extended periods of time toanalyse whether myeloid leukaemia or myelo-proliferative disorders woulddevelop. So far 8 mice were reconstituted at 6-8 weeks of age andanalysed at 8, 11, 14 and 22 months of age, the latter of which is closeto the maximal life span of a mouse. During the whole observation periodthe mice appeared normal and showed no sign of discomfort. Regular bloodanalysis up to 22 months showed normal white blood cell counts andmorphology without indication of blasts or abnormal cells. By FACSanalysis monocytes had a normal CD115+, CD11b+, F4/80+, phenotype and noincreased numbers of cells expressing immature or progenitor surfacemarkers were detected. Upon sacrifice, dissection also did not revealany macroscopic signs of leukemia, the spleen presented a normal sizeand healthy appearance. FACS analysis and histological staining ofcytospins from blood, bone marrow, spleen and peritoneal exudate did notreveale any indication of leukaemia or myelo-proliferative disease. Noincreased numbers of cells with a progenitor surface marker profile(CMP,GMP) or cells with blast or immature morphology were observed inthe bone marrow. This indicates that c-Maf/MafB deficient monocytes andmonocyte derived cells can contribute normally and long term to thehematopoietic system without causing hematologic pathologies.

c-Maf/MafB deficient monocytes from aged mice can be expanded in M-CSFculture: We wanted to analyze whether c-Maf/MafB deficientmonocyte/macrophages with expanded self-renewal capacity could not onlybe derived from embryonic cells but also from terminally differentiatedcells of adult or even aged animals. Therefore we tested whethermonocytes from mice reconstituted with a MafB/c-Maf deficienthematopoietic system could be expanded in M-CSF culture and give rise tocolonies in M-CSF containing methocell medium. As shown in FIG. 6,monocytes from wt reconstituted mice could not give rise to colonies inM-CSF methocell culture, as was expected. By contrast, c-Maf/MafBdeficient white blood cells gave rise to CFU-M monocytic colonies andcould be expanded in M-CSF culture. Together this demonstrated that theobserved phenotype is not specific to embryonic cells but thatMafB/c-Maf deficiency confers extended self-renewal capacity toterminally differentiated monocytes from adult aged mice.

c-Maf/MafB deficient monocyte/macrophage expanded in culture home toperipheral tissues after intravenous injection: To test whether in M-CSFculture expanded c-Maf/MafB cells would emigrate from the circulationinto peripheral tissues like normal monocytes, we reinjected 1×10⁶Ly5.2+ c-Maf/MafB deficient monocyte/macrophages intravenously intoLy5.1+ hosts and analysed their presence in the circulation and inperipheral tissues after 4, 24 and 48 hours. We observed that CD11b+donor cells were detectable in the peritoneum from 4 hours on and in thespleen from 24 h on. This indicated that in culture expanded c-Maf/MafBcells could contribute to monocyte derived cell populations in vivo.

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1. An ex vivo method for expanding monocytes, macrophages or dendriticcells, which method comprises inhibiting the expression or the activityof MafB and c-Maf in monocytes, macrophages or dendritic cells; andexpanding the cells in the presence of at least one cytokine.
 2. Themethod according to claim 1, wherein the expression of MafB and c-Maf isinhibited by using siRNA oligonucleotide, antisense oligonucleotide orribozymes.
 3. The method according to claim 1, wherein the activity ofMafB and c-Maf is inhibited by using mutated MafB and c-Maf polypeptideswhich compete with the wild-type MafB and c-Maf.
 4. The method accordingto claim 1, wherein the cytokine is M-CSF.
 5. (canceled)
 6. A monocyte,macrophage or dendritic cell, which does not express MafB and c-Maf orin which the expression or activity of MafB and c-Maf is abolished orinhibited.
 7. A monocyte, macrophage or dendritic cell of claim 6, whichlacks the MafB and c-Maf genes.
 8. A monocyte, macrophage or dendriticcell according to claim 6, which is of murine origin.
 9. A monocyte,macrophage or dendritic cell according to claim 6, which is of humanorigin.
 10. A macrophage according to claim 6, which is selected fromthe group consisting of microglia, histiocytes, Hofbauer cells,mesangial cells, Kupffer cells, peritoneal macrophages, alveolarmacrophage, epidermal or dermal macrophages, marginal zone macrophages,metallophilic macrophages, Red pulp macrophages, white pulp macrophagesand osteoclasts.
 11. A dendritic cell according to claim 6, which isfurther loaded with an antigenic molecule.
 12. A pharmaceuticalcomposition comprising a monocyte, macrophage or dendritic cellaccording to claim 6, in combination with a pharmaceutically acceptablecarrier.
 13. A method of vaccination of an animal comprisingadministering a pharmaceutical composition which comprises the dendriticcell of claim 11, loaded with an antigenic molecule, in an amounteffective for use as a vaccine.
 14. A method for the treatment of adisease selected from the group consisting of a cancer, acute oracquired immuno-deficiencies, chronic or acute injury, wounds,degenerative diseases, autoimmune diseases, chronic inflammatorydiseases, atherosclerosis, poly- and osteo-artritis, osteoporosis,infectious diseases, and metabolic diseases comprising administration ofa monocyte, macrophage or dentritic cell according to claim
 6. 15. Amethod for regenerative medicine comprising administration of amonocyte, macrophage or dendritic cell according to claim
 6. 16. Amethod for the screening of drugs comprising the use of a monocyte,macrophage or dendritic cell according to claim
 6. 17. A method forgenerating and expanding murine monocytes, which method comprises thesteps consisting of: i) isolating monocytes derived from a mouse whichdoes not express MafB and c-Maf and ii) culturing said monocytes in thepresence of M-CSF.
 18. A monocyte, macrophage or dentritic cellaccording to claim 6, wherein said monocyte, macrophage or dentriticcell is obtained by the method for expanding said monocytes, macrophagesor dendritic cells, which method comprises inhibiting the expression orthe activity of MafB and c-Maf in monocytes, macrophages or dendriticcells; and expanding the cells in the presence of at least one cytokine.